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- PDB-7ujj: Stx2a and DARPin complex -

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Basic information

Entry
Database: PDB / ID: 7ujj
TitleStx2a and DARPin complex
Components
  • (Shiga-like toxin 2 subunit ...) x 2
  • DARPin
KeywordsANTITOXIN / DARPin / shiga toxin
Function / homology
Function and homology information


symbiont-mediated modulation of host virulence / hemolysis by symbiont of host erythrocytes / rRNA N-glycosylase / rRNA N-glycosylase activity / toxin activity / negative regulation of translation / extracellular region
Similarity search - Function
Shiga-like toxin, subunit A / Shiga-like toxin, beta subunit / Shiga-like toxin beta subunit / Enterotoxin / Ribosome-inactivating protein conserved site / Shiga/ricin ribosomal inactivating toxins active site signature. / Ribosome-inactivating protein / Ribosome-inactivating protein, subdomain 1 / Ribosome-inactivating protein, subdomain 2 / Ribosome-inactivating protein superfamily / Ribosome inactivating protein
Similarity search - Domain/homology
3-PYRIDINIUM-1-YLPROPANE-1-SULFONATE / FORMIC ACID / Shiga-like toxin 2 subunit A / Shiga-like toxin 2 subunit B
Similarity search - Component
Biological speciesSynthetic construct (others)
Escherichia phage 933W (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsJiang, M. / Zhang, J.
Funding support United States, 1items
OrganizationGrant numberCountry
Welch FoundationA1863 United States
CitationJournal: Bioengineering (Basel) / Year: 2022
Title: A Multi-Specific DARPin Potently Neutralizes Shiga Toxin 2 via Simultaneous Modulation of Both Toxin Subunits.
Authors: Yu Zeng / Mengqiu Jiang / Sally Robinson / Zeyu Peng / Vikas Chonira / Rudo Simeon / Saul Tzipori / Junjie Zhang / Zhilei Chen /
Abstract: Shiga toxin-producing (STEC) is a common cause of bloody diarrhea. The pathology of STEC infection derives from two exotoxins-Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2)-that are secreted by STEC ...Shiga toxin-producing (STEC) is a common cause of bloody diarrhea. The pathology of STEC infection derives from two exotoxins-Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2)-that are secreted by STEC in the gut, from where they are systemically absorbed, causing severe kidney damage leading to hemolytic uremic syndrome (HUS). Currently, there is no effective treatment for HUS, and only supportive care is recommended. We report the engineering of a panel of designed ankyrin repeat proteins (DARPin) with potent neutralization activity against Stx2a, the major subtype associated with HUS. The best dimeric DARPin, SD5, created via a combination of directed evolution and rational design, neutralizes Stx2a with a half maximal effective concentration (EC) of 0.61 nM The two monomeric DARPin constituents of SD5 exhibit complementary functions-SHT targets the enzymatic A subunit of Stx2a and inhibits the toxin's catalytic activity, while DARPin #3 binds the B subunit, based on the cryo-EM study, and induces a novel conformational change in the B subunit that distorts its five-fold symmetry and presumably interferes with toxin attachment to target cells. SD5 was fused to an albumin-binding DARPin, and the resulting trimeric DARPin DA1-SD5 efficiently protects mice in a toxin challenge model, pointing to a high potential of this DARPin as a therapeutic for STEC infection. Finally, the unprecedented toxin conformational change induced by DARPin #3 represents a novel mode of action for neutralizing Stx2 toxicity and reveals new targets for future drug development.
History
DepositionMar 30, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 12, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: DARPin
A: Shiga-like toxin 2 subunit A
B: Shiga-like toxin 2 subunit B
C: Shiga-like toxin 2 subunit B
D: Shiga-like toxin 2 subunit B
E: Shiga-like toxin 2 subunit B
F: Shiga-like toxin 2 subunit B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)94,13536
Polymers92,2157
Non-polymers1,92129
Water9,206511
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 1 molecules G

#1: Protein DARPin


Mass: 19863.410 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synthetic construct (others)
Production host: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)

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Shiga-like toxin 2 subunit ... , 2 types, 6 molecules ABCDEF

#2: Protein Shiga-like toxin 2 subunit A / SLT-2 A subunit / SLT-2a / SLT-IIa / Verocytotoxin 2 subunit A / Verotoxin 2 subunit A / rRNA N-glycosidase 2


Mass: 33228.215 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage 933W (virus) / Gene: stxA2, stx2A, L0103
Production host: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
References: UniProt: P09385, rRNA N-glycosylase
#3: Protein
Shiga-like toxin 2 subunit B / SLT-2 B subunit / SLT-2b / SLT-IIb / Verocytotoxin 2 subunit B / Verotoxin 2 subunit B


Mass: 7824.590 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage 933W (virus) / Gene: stxB2, stx2B, L0104
Production host: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
References: UniProt: P09386

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Non-polymers , 5 types, 540 molecules

#4: Chemical
ChemComp-1PS / 3-PYRIDINIUM-1-YLPROPANE-1-SULFONATE / 1-(3-SULFOPROPYL) PYRIDINIUM / PPS


Mass: 201.243 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H11NO3S
#5: Chemical
ChemComp-FMT / FORMIC ACID


Mass: 46.025 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: CH2O2
#6: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Na
#7: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C2H6O2
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 511 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Ternary complex of stx2 and DARPinCOMPLEX#1-#30MULTIPLE SOURCES
2Shiga-like toxin 2 subunit ACOMPLEX1RECOMBINANT
3Shiga-like toxin 2 subunit BCOMPLEX2RECOMBINANT
4DARPinCOMPLEX3RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia phage 933W (virus)10730
31Escherichia phage 933W (virus)10730
41Synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)1182032
33Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)1182032
44Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)1182032
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 108744 / Symmetry type: POINT

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